Intercellular fusion is fundamental to the conception, development and physiology of multicellular organisms. Compared with vesicle fusion and virus-cell fusion, relatively little is known of the mechanistic underpinnings of cell-cell fusion. A mechanistic understanding of cell-cell fusion is not only important for fundamental biology but may also provide basis for its manipulation in therapeutic settings. With the long-term goal of understanding the mechanisms underlying cell-cell fusion, we have reconstituted cell fusion by co-expressing a Drosophila cell adhesion molecule Sns and a C. elegans fusogen Eff-1 in a fly cell line that otherwise does not fuse. Our studies using this culture system have revealed a general function for actin-propelled membrane protrusions in promoting fusogenic protein engagement at the site of fusion, known as the fusogenic synapse. This culture system also provides a great platform for gain-of-function and loss-of-function screens for new components of cell-cell fusion, which are otherwise difficult to uncover by loss-of-function genetic screens due to maternal contributions and functional redundancy. Moreover, this culture system makes biophysical manipulations of cell-cell fusion possible, which is technically challenging in an intat organism. Our recent functional screens using this culture system led to the identification of cholesterol-efflux ATP-binding cassette (ABC) transporters in cell-cell fusion, opening up an exciting new research direction in the field. We will elucidate the molecular function of ABC transporters in S2R+ cell fusion by testing whether the Drosophila ABC transporters function as conventional ABC transporters; whether these ABC transporters specifically transport cholesterol; and whether these ABC transporters regulate cholesterol distribution at the fusogenic synapse. Our recent studies have also identified a role for membrane skeletal protein Spectrin in cell-cell fusion. Spectrin co-localizes with MyoII at the fusogenic synapse and is ideally positioned as a mechanotransducer that links the plasma membrane to the actomyosin network. Moreover, Spectrin is required for restricting the localization domain of the cell adhesion molecule Duf. We will explore how Spectrin restricts Duf localization using a combined approach including biochemistry, genetics and super-resolution imaging. We will also investigate the potential function of Spectrin in mechanotransduction during cell-cell fusion using genetic, cell biological and biophysical methods. These proposed studies promise to bring novel mechanistic insights into the function of lipids, especially cholesterol, and membrane-cytoskeleton mechanotransduction during intercellular fusion.